Hydrolysis of Extracellular ATP by Ectonucleoside Triphosphate Diphosphohydrolase (ENTPD) Establishes the Set Point for Fibrotic Activity of Cardiac Fibroblasts

The establishment of set points for cellular activities is essential in regulating homeostasis. Here, we demonstrate key determinants of the fibrogenic set point of cardiac fibroblasts (CFs) by focusing on the pro-fibrotic activity of ATP, which is released by CFs. We tested the hypothesis that the hydrolysis of extracellular ATP by ectonucleoside triphosphate diphosphohydrolases (ENTPDs) regulates pro-fibrotic nucleotide signaling. We detected two ENTPD isoforms, ENTPD-1 and -2, in adult rat ventricular CFs. Partial knockdown of ENTPD-1 and -2 with siRNA increased basal extracellular ATP concentration and enhanced the pro-fibrotic effect of ATP stimulation. Sodium polyoxotungstate-1, an ENTPD inhibitor, not only enhanced the pro-fibrotic effects of exogenously added ATP but also increased basal expression of α-smooth muscle actin, plasminogen activator inhibitor-1 and transforming growth factor (TGF)-β, collagen synthesis, and gel contraction. Furthermore, we found that adenosine, a product of ATP hydrolysis by ENTPD, acts via A2 B receptors to counterbalance the pro-fibrotic response to ATP. Removal of extracellular adenosine or inhibition of A2 B receptors enhanced pro-fibrotic ATP signaling. Together, these results demonstrate the contribution of basally released ATP in establishing the set point for fibrotic activity in adult rat CFs and identify a key role for the modulation of this activity by hydrolysis of released ATP by ENTPDs. These findings also imply that cellular homeostasis and fibrotic response involve the integration of signaling that is pro-fibrotic by ATP and anti-fibrotic by adenosine and that is regulated by ENTPDs. Background: Ectonucleoside triphosphate diphosphohydrolases (ENTPDs) hydrolyze extracellular ATP and diminish ATP signaling. Results: ENTPD inhibition increases the fibrotic phenotype of cardiac fibroblasts (CFs) by enhancing pro-fibrotic response to ATP and attenuating anti-fibrotic response to adenosine. Conclusion: ENTPDs integrate ATP and adenosine signaling to regulate the CF phenotype. Significance: ENTPD is an important regulatory component controlling ATP- and adenosine-mediated CF homeostasis.